A scientist at the Marine Biological
Laboratory (MBL) has published the results of an EPA-funded clam embryo
study that supports her hypothesis that, when combined, the pollutants
bromoform, chloroform, and tetrachloroethylene--a chemical cocktail
known as BCE--can act synergistically to alter a key regulator in nerve
cell development. While scientists have previously studied the effects
of these pollutants individually, this is the first time anyone has
demonstrated that BCE's components can work together to adversely
affect neuron growth in a model organism.
The study, which is reported in the January 2005 issue of Environmental
Toxicology and Pharmacology, is the first step toward understanding how
exposure to BCE might affect human nerve cell development--knowledge
that may one day provide clues about such neurological mysteries as
autism spectrum disorder or attention deficit hyperactivity disorder.

To
test her hypothesis, MBL scientist Carol Reinisch and her colleagues
treated developing surf clam embryos (Spisula solidissima) with
different combinations of BCE and studied their effects on nerve cell
growth. "On a cellular level, clam neurons are extremely useful in
studying basic mechanisms of cell development," says Reinisch, an
expert in PCB-induced neurotoxicity.
"Of the different combinations and strengths of BCE components tested,
we found that all three together induce the greatest adverse response.
Treating the embryos with the triple mixture resulted in increased
production of a subunit of an enzyme called protein kinase A (PKA),
which previous research suggests plays a role in neural development,"
says Reinisch. "Fluctuations in PKA may influence not only neuronal
maturation but also how neuronal networks are constructed during
development," she says. Alterations of this enzyme may affect neural
development and neural connections by activating or inactivating other
proteins.
Demonstrating that clam embryos are affected by BCE paves the way for
additional studies that may help explain how exposure to BCE affects
human nerve cell development and how it might relate to neurological
disorders. "We can clearly state that we found an increase in a
component of PKA, and PKA is known to be involved in neural
development. The BCE mixture is capable of altering neural development,
and alterations in neural development are thought to be a cause of
autism," says Jill Kreiling, first author of the paper and a member of
Reinisch's lab. But Kreiling cautions, "We cannot say at this time if
the alteration we see in clam embryos is the same alteration that
causes autism. That will require future research."
In fact, Reinisch and Kreiling have already begun the next phase of
their work. They are currently examining the origins of BCE toxicity at
the single-neuron level to learn what genes are turned on and off by
the exposure to the chemical mixture. The research is focused on a
family of genes known as P53, which helps to regulate the cell cycle.
They have also moved their studies to a new model system: the
zebrafish, a vertebrate with more similarities to humans.
Reinisch's work on BCE is funded by a STAR grant from the United States
Environmental Protection Agency. Science supported by the STAR program
is rigorously peer reviewed.
Study Background and Research Methods
When MBL researcher Carol Reinisch first heard news stories about Brick
Township, New Jersey--a town that was the center of an ATSDR* study in
the year 2000 that documented a higher than usual rate of autism
spectrum disorder and explored its link to bromoform, chloroform, and
tetrachloroethylene in Brick's water supply--it got her thinking. "My
hypothesis was this: While it had been proven that individually the
chemicals don't impact nerve cell development, the combination of all
three might pose increased risks," she says.
So Reinisch decided to test the chemical cocktail, in similar
proportions to those found in Brick, on a species of surf clam readily
available in the waters off of her Cape Cod lab. The species is famous
among scientists as a model for the study of cellular processes that
are common among all animals, including humans.
Reinisch and her colleagues collected surf clam eggs and sperm,
fertilized them in vitro, and added BCE to normally developing clam
embryos. The investigators used a combination of antibodies and dyes to
show the presence of neurotransmitters in single neurons. "We've
actually taken superlative three-dimensional pictures where we go right
through the embryo," says Reinisch. The sequence of images yields a
three-dimensional representation of the embryos' developing neurons.
Reinisch and company examined periodically recorded images to see what
effect, if any, the chemicals had. They also measured the levels of
proteins in the normal and treated embryos to check the visual data. Read the full paper'"/>

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